RNA interference (RNAi) has become one of the most powerful and widely used research tools in molecular biology and functional genomics. There is also an intriguing potential that RNAi may become a new therapeutic approach. Despite the remarkable progress, RNAi is far from being a perfect tool and needs significant improvement for in vivo research and therapeutic applications. In particular, the potency, delivery and cellular uptake, biodistribution, and enzymatic stability of short interfering RNAs (siRNAs) must be improved. The long-term goal of our research is to develop chemical modifications that optimize the properties of siRNAs while studying structure and function of RNA in a broader context. The present renewal builds on our discoveries, made during the first period of this grant, that amide is an excellent structural replacement for phosphate in RNA. The intellectual framework of the renewal builds around broad hypotheses that amides may also mimic the phosphate-amino acid interactions in RNA-protein complexes and may significantly improve the properties of siRNAs. The central innovative aspect of this proposal is in replacing the negatively charged and polar phosphate with the neutral and relatively hydrophobic amide linkage. Such a dramatic modification of RNA's backbone has little precedent in the RNAi field and, if successful, will initiate a paradigm shift in our thinking of what can and what cannot be tolerated in siRNAs. Reduction of siRNA's charge will allow design of novel siRNA-cell penetrating peptide conjugates, which is not possible with unmodified RNA. The preliminary results strongly support our hypotheses that replacement of phosphates with amides will be well tolerated in siRNAs. Thus, the proposal will challenge the current paradigm that negatively charged phosphates are required for recognition of RNA by proteins. The specific aims will 1) study the RNAi activity of amide-modified siRNAs; 2) study the recognition of amide-modified RNA by the Piwi domain of Argonaute protein; and 3) improve the cellular uptake using novel siRNA-cell penetrating peptide conjugates. The aims will test specific hypotheses relevant to molecular recognition of modified siRNAs and broader RNAi mechanism. The aims will be achieved through a comprehensive and multidisciplinary study involving collaborative efforts in synthetic and biophysical chemistry (PI), structural biochemistry (Egli, crystallography and Kennedy, NMR), RNA biology (Thermo Fisher), and cell biochemistry and fluorescence microscopy (Grewer and McGee). The main impact of successfully reaching the proposed aims will be improved chemically modified siRNAs for in vivo applications as research tools and, potentially, as lead compounds for drug development. The ultimate goal is to solve the delivery and cellular uptake problems, which would dramatically expand the ability to use RNAi in animals to study physiology of disease. The proposed studies will also advance fundamental knowledge on RNA-protein interactions and provide unique insights into RNAi mechanism.